Cd4 bv786

PBMC were collected from the patients’ EDTA blood samples using a lymphocyte separation medium (Ficoll-Human density 1.077g/mL, PAN Biotech, Aidenbach, Germany). Harvested cells were cryopreserved in a 9:1 medium of fetal calf serum (Gibco) and dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) until immunophenotyping analysis.
Lymphocyte subpopulations were determined with the following panel of 14 antibodies (BD Biosciences, Franklin Lakes, NJ, USA): CD3-APC-H7, CD4-BV786, CD8α-BV711, CD19-BV510, CD25-BV421, CD45RA-PE-Cy7, CD56-BV605, CD127-AF647, CD183-BB700, CD194-BV650, CD196-PE-CF594, CD197-BB515, Foxp3-PE and a viability marker (FVS-APC R700). Frozen PBMC were thawed, and a total of 500,000 cells were labeled after saturation with human serum. Cells were successively stained with the viability dye, the surface antibodies and, finally, with FoxP3 after cell fixation and permeabilization with Transcription Factor Buffer (BD Biosciences). For all experiments, acquisition was performed using LSR-Fortessa flow cytometer (BD Biosciences) and data were analyzed using Flowjo software (version 10). The gating strategy is detailed in the Supplementary Data section (Supplementary Figure S1). In order to avoid uncertain results, the TCD4 and TCD8 subpopulations were not analyzed when there were fewer than 500 events for TCD4 or TCD8 cells.

For surface staining, antibodies were diluted in brilliant stain buffer (BD Biosciences, P/N 563794) and cells were stained with this mixture. Washing steps were performed with a standard flow cytometry buffer (1% FBS, 0.02% NaN₃, PBS). For intracellular staining, the Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, P/N 00-5523-00) was used according to the manufacturer’s instructions. Cells were pretreated with GolgiStop for cytokine staining (BD, P/N 554724). Viability was assessed using either 7-AAD (BioLegend, P/N 420403) or fixable viability dye eFluor780 (Invitrogen, P/N 65-0865-14). All antibodies used were from BD Biosciences: CD3-PE (P/N 566683), CD4-FITC (P/N 300538), CD4-BV786 (P/N 563877), IFN-γ-FITC (P/N 554551), Isotype control-FITC (P/N 554679), CD9-PE (P/N 555372), CD30-BV421 (P/N 562876), CD45RA-BV711 (P/N 612847), CD45RO-BV421 (P/N 562641), CD52-AF647 (P/N 563610), CD96-BV711 (P/N 563174).

Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).